Most bacteria polymerize peptidoglycan (PG) into a mesh-like sacculus that surrounds the cytoplasmic membrane and protects it against osmotic lysis. The PG sacculus also provides cell shape and serves as a scaffold to which virulence factors are anchored. Biogenesis of the PG sacculus is essential for viability, and our long-term goal is to understand it at the molecula level. This proposal focuses on the transport of PG intermediates across the cytoplasmic membrane, a poorly understood step in PG biogenesis. Bacteria build their PG sacculus in the extracytoplasmic space by polymerizing a disaccharide pentapeptide into glycan strands that are later crosslinked. The disaccharide pentapeptide is made in the cytoplasm as a lipid intermediate known as lipid II. Therefore, lipid II must be flipped across the membrane by a transporter (a flippase) through an unknown mechanism. The membrane protein MurJ has been proposed to be the lipid II flippase in Escherichia coli since it is essential for PG synthess and it belongs to the MOP (multidrug/oligosaccharidyl- lipid/polysaccharide) superfamily of exporters. This family is conserved in diverse organisms and it includes flippases of lipid-linked oligosaccharides that are similar to lipid II. FtsW and RodA have also been proposed to be lipid II flippases in E. coli, but there is no in vivo evidence supporting this hypothesis. Aim 1 of thi proposal is to determine whether MurJ, FtsW, and RodA are involved in lipid II translocation in vivo. Two analytical assays based on differential labeling and mass spectrometry will be developed to assess how depletion of these factors affects the levels and membrane topology of lipid II. The data obtained will be crucial for understanding the roles of MurJ, FtsW, and RodA in PG biogenesis. Aim 2 of this proposal is to determine how MurJ functions. Structural information will be obtained by determining the membrane topology of MurJ. Functional partners of MurJ will be identified biochemically and genetically. Mutation and suppression analyses will be conducted to identify domains of MurJ required for stability, intra- and inter- molecular interactions, and activity. Together, the data obtained will uncover the essential function of MurJ in PG biogenesis. Many of the most effective antibiotics target PG biogenesis. MurJ and other proteins required for PG biogenesis are potential targets for antibiotics since PG is essential in most bacteria and is absent in humans. The proposed work will aid in the development of MurJ inhibitors, which may prove to be much-needed novel antibiotics. In addition, understanding MurJ function will advance knowledge of the MOP superfamily of exporters, which includes members that are involved in bacterial pathogenesis and certain types of human congenital disorders of glycosylation.